The major objective of this application is to develop and optimize a unique yeast system for systematic identification and characterization of host factors of viral genome replication and gene expression in nonsegmented negative-strand (NNS) RNA viruses (taxonomic order Mononegavirales). This order contains a wide variety of human and animal viruses, including many lethal human pathogens. The viral RNA polymerase of NNS RNA viruses consists of two viral subunits, and includes a number of largely unidentified host protein factors. Our current understanding of the contributions of host proteins in viral replication is very limited, and there is an urgent need to develop new systematic approaches to the identification of host factors, which should serve as promising antiviral targets. The proposed pilot experiments will be focused on a prototypic Mononegavirales, vesicular stomatitis virus (VSV, a rhabdovirus). The first aim will test the feasibility of the yeast Saccharomyces cerevisiae as an experimental host supporting genome replication and gene expression of VSV. If it works, this system should be suitable for efficient, cost-effective, and high-throughput systematic identification of host protein factors important for viral RNA synthesis in VSV and other Mononegavirales. The second aim will employ a newly-developed "cytoplasmic" yeast 2-hybrid system for the identification of cellular proteins physically interacting with VSV proteins. The identification of host factors of virus RNA replication and transcription should provide new clues for understanding the molecular mechanisms of viral RNA synthesis and for developing new ways to combat viral infections via control of host components. PUBLIC HEALTH RELEVANCE: The major objective of this application is to develop a new yeast-based experimental system to study virus- host interactions in nonsegmented negative-strand RNA viruses (taxonomic order Mononegavirales). This order contains many lethal human pathogens, including the Ebola and Marburg viruses which are classified as category A bioweapon agents by the Centers for Disease Control and Prevention, and highly prevalent human pathogens, such as the respiratory syncytial and parainfluenza viruses. Although infections caused by many Mononegavirales have been largely controlled in the developed world through a variety of vaccination and therapeutic strategies, they still cause many clinically devastating diseases (including many emerging diseases), which contribute significantly to world-wide morbidity and mortality. It continues to be important to define the basic mechanisms of reproduction of these viruses, especially virus-host interactions, with the ultimate hope that this knowledge will contribute not only to the basic understanding of virus biology, but will also lead to new or better therapeutic agents. [unreadable] [unreadable] [unreadable]